Regulation of gene expression by RNA structure occurs in both prokaryotic and eukaryotic organisms. One common mode of RNA based regulation in bacteria is riboswitch control. This type of gene regulation depends on the ability of one RNA sequence to form two different structures. Therefore, RNA conformational change is crucial to riboswitch regulation. Small molecule metabolite binding to an aptamer domain in nascent mRNA during transcription controls the final structure formed by that RNA. One structure represses the encoded proteins by terminating transcription or preventing translation. The alternative structure allows gene expression. One such riboswitch is the vitamin B12 btuB riboswitch of E. coli. This RNA controls the expression of an outer membrane B12 transport protein in response to the cellular concentration of vitamin B12. To accomplish this the same RNA sequence must form an 'on' or an 'off' structure dependent on B12 binding to the RNA. Traditionally, biophysical methods are used to study the formation and rearrangement of RNA structure in completed transcripts. However, riboswitch regulation occurs during transcription. To understand the mechanism of riboswitch function, folding and rearrangement will be studied during transcription. The benefit of this research is twofold. First it will provide a mechanistic understanding of the btuB system and insight into riboswitch based gene regulation in bacteria. Riboswitches are widespread genetic control elements. Second, the proposal will lay a general conceptual framework for the understanding of RNA conformational change during transcription. PUBLIC HEALTH RELEVANCE: This research will provide a mechanistic understanding of riboswitch function in bacteria by studying RNA folding during transcription, the same way it occurs in cells. This information is expected to give insight into the regulation of key biochemical pathways in human pathogens. It will also give a conceptual framework and new tools for understanding the mechanisms of more complex, non-coding RNA based regulatory systems in higher organisms.